Ganoderma lucidum extract attenuates corticotropin-releasing hormone-induced cellular senescence in human hair follicle cells.

Corticotropin-releasing hormone (CRH) is a key mediator in stress-induced hair growth inhibition. Here, we investigated the impact of stress-induced senescence and evaluated the potential of Ganoderma lucidum (GL) extract in mitigating CRH-induced senescence in human hair follicle cells (hHFCs). We show that CRH treatment increased the senescence-associated beta-galactosidase (SA-ß-GAL) activity and reactive oxygen species (ROS) formation in hHFCs and suppressed alkaline phosphatase (ALP) activity and anagen-inducing genes. However, GL extract restored ALP activity and decreased the expression levels of anagen-related genes in CRH-treated hHFCs. It decreased SA-ß-GAL activity, reduced ROS production, and prevented the phosphorylation of MAPK signaling pathways in CRH-related stress response. Moreover, GL reversed the CRH-induced inhibition of two-cell assemblage (TCA) elongation and Ki67 expression. GL extract attenuates stress-induced hair follicular senescence by delaying catagen entry and scavenging ROS. Our findings suggest that GL extract could be used for treating stress-induced hair loss.

Inhibition of class I HDACs preserves hair follicle inductivity in postnatal dermal cells.

Induction of new hair follicles (HFs) may be an ultimate treatment goal for alopecia; however, functional cells with HF inductivity must be expanded in bulk for clinical use. In vitro culture conditions are completely different from the in vivo microenvironment. Although fetal and postnatal dermal cells (DCs) have the potential to induce HFs, they rapidly lose this HF inductivity during culture, accompanied by a drastic change in gene expression. This suggests that epigenetic regulation may be involved. Of the various histone deacetylases (HDACs), Class I HDACs are noteworthy because they are ubiquitously expressed and have the strongest deacetylase activity. This study revealed that DCs from postnatal mice rapidly lose HF inductivity and that this reduction is accompanied by a significant decrease in histone H3 acetylation. However, MS-275, an inhibitor of class I HDACs, preserves HF inductivity in DCs during culture, increasing alkaline phosphatase activity and upregulating HF inductive genes such as BMP4, HEY1, and WIF1. In addition, the inhibition of class I HDACs activates the Wnt signaling pathway, the most well-described molecular pathway in HF development, via increased histone H3 acetylation within the promoter region of the Wnt transcription factor LEF1. Our results suggest that class I HDACs could be a potential target for the neogenesis of HFs.

"Two-Cell Assemblage" Assay: A Simple in vitro Method for Screening Hair Growth-Promoting Compounds.

Alopecia arises due to inadequate hair follicle (HF) stem cell activation or proliferation, resulting in prolongation of the telogen phase of the hair cycle. Increasing therapeutic and cosmetic demand for alleviating alopecia has driven research toward the discovery or synthesis of novel compounds that can promote hair growth by inducing HF stem cell activation or proliferation and initiating the anagen phase. Although several methods for evaluating the hair growth-promoting effects of candidate compounds are being used, most of these methods are difficult to use for large scale simultaneous screening of various compounds. Herein, we introduce a simple and reliable in vitro assay for the simultaneous screening of the hair growth-promoting effects of candidate compounds on a large scale. In this study, we first established a 3D co-culture system of human dermal papilla (hDP) cells and human outer root sheath (hORS) cells in an ultra-low attachment 96-well plate, where the two cell types constituted a polar elongated structure, named "two-cell assemblage (TCA)." We observed that the long axis length of the TCA gradually increased for 5 days, maintaining biological functional integrity as reflected by the increased expression levels of hair growth-associated genes after treatment with hair growth-promoting molecules. Interestingly, the elongation of the TCA was more prominent following treatment with the hair growth-promoting molecules (which occurred in a dose-dependent manner), compared to the control group (p < 0.05). Accordingly, we set the long axis length of the TCA as an endpoint of this assay, using a micro confocal high-content imaging system to measure the length, which can provide reproducible and reliable results in an adequate timescale. The advantages of this assay are: (i) it is physiologically and practically advantageous as it uses 3D cultured two-type human cells which are easily available; (ii) it is simple as it uses length as the only endpoint; and (iii) it is a high throughput system, which screens various compounds simultaneously. In conclusion, the "TCA" assay could serve as an easy and reliable method to validate the hair growth-promoting effect of a large volume of library molecules.

Caffeoyl-Prolyl-Histidine Amide Inhibits Fyn and Alleviates Atopic Dermatitis-Like Phenotypes via Suppression of NF-κB Activation.

Caffeic acid (CA) is produced from a variety of plants and has diverse biological functions, including anti-inflammation activity. It has been recently demonstrated that caffeoyl-prolyl-histidine amide (CA-PH), which is CA conjugated with proline-histidine dipeptide, relieves atopic dermatitis (AD)-like phenotypes in mouse. In this study, we investigated the molecular mechanism underlying CA-PH-mediated alleviation of AD-like phenotypes using cell line and AD mouse models. We confirmed that CA-PH suppresses AD-like phenotypes, such as increased epidermal thickening, infiltration of mast cells, and dysregulated gene expression of cytokines. CA-PH suppressed up-regulation of cytokine expression through inhibition of nuclear translocation of NF-κB. Using a CA-PH affinity pull-down assay, we found that CA-PH binds to Fyn. In silico molecular docking and enzyme kinetic studies revealed that CA-PH binds to the ATP binding site and inhibits Fyn competitively with ATP. CA-PH further suppressed spleen tyrosine kinase (SYK)/inhibitor of nuclear factor kappa B kinase (IKK)/inhibitor of nuclear factor kappa B (IκB) signaling, which is required for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. In addition, chronic application of CA-PH, in contrast with that of glucocorticoids, did not induce up-regulation of regulated in development and DNA damage response 1 (REDD1), reduction of mammalian target of rapamycin (mTOR) signaling, or skin atrophy. Thus, our study suggests that CA-PH treatment may help to reduce skin inflammation via down-regulation of NF-κB activation, and Fyn may be a new therapeutic target of inflammatory skin diseases, such as AD.

Caffeoyl-Pro-His amide relieve DNCB-Induced Atopic Dermatitis-Like phenotypes in BALB/c mice.

The main factors involved in the pathogenesis of atopic dermatitis (AD) are skin barrier abnormality, allergy/immunology, and pruritus. Considering how oxidative stress influences these factors, antioxidant agents may be effective candidates in the treatment of AD. To evaluate the effect of Caffeoyl-Pro-His amide (CA-PH), an antioxidant agent, on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like phenotypes in BALB/c mice. Topical sensitization and challenge by DNCB were performed on the dorsal skin of BALB/c mice to induce AD-like cutaneous lesions, phenotypes, and immunologic response. CA-PH was applied topically for 2 weeks to assess its effects on DNCB-induced AD-like phenotypes. As a result, CA-PH relieved DNCB-induced AD-like phenotypes quantified by dermatitis severity score, scratching duration, and trans-epidermal water loss. Histopathological analysis showed that CA-PH decreased epidermal thickening, the number of mast cells, and eosinophil infiltration in dermis. Immunohistochemical staining revealed that CA-PH recovered skin barrier-related proteins: filaggrin, involucrin, and loricrin. As for the immunologic aspects, CA-PH treatment lowered mRNA or protein levels of interleukin (IL)-4, IL-6, IL-17a, IL-1b, IL-31, and IL-33 levels and thymic stromal lymphopoietin (TSLP) levels in cutaneous tissue, reducing the DNCB-induced serum IgE level elevation. In conclusion, topical CA-PH may be a therapeutic option for the treatment of AD.

IL-17A-Producing Innate Lymphoid Cells Promote Skin Inflammation by Inducing IL-33-Driven Type 2 Immune Responses.

Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disease characterized by type 2 cytokines secreted by T helper type 2 cells and group 2 innate lymphoid cells. Despite a high degree of heterogeneity, AD is still explained by type 2 immunity, and the role of IL-17A, which is increased in acute, pediatric, or Asian patients with AD, remains poorly understood. Here, we aimed to investigate the role of IL-17A-producing group 3 innate lymphoid cells (ILC3s), which are unexplored immune cells, in the pathogenesis of AD. We found that the numbers of ILC3s in the skin of AD-induced mice were increased, and that neutralizing IL-17A delayed development of AD. Moreover, adoptive transfer of ILC3s accelerated the symptoms of AD. Mechanically, ILC3s induced IL-33 production by nonimmune skin cells, keratinocytes, and fibroblasts, which promoted type 2 immune responses. Because AD has a complex pathophysiology and a broad spectrum of clinical phenotypes, the presence of ILC3s in the skin and their interaction with nonimmune skin cells could explain the pathogenesis of cutaneous AD.

Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle.

Shikimic acid (SA) has recently been found to be a major component of plant stem cells. The exact effects of SA on human hair follicles (HFs) is unknown. The purpose of this study was to examine the effects of SA on hair growth. We investigated the effect of SA on an in vivo C57BL/6 mouse model. We examined the expression of mannose receptor (MR), which is a known receptor of SA, in human HFs and the effect of SA on human dermal papilla cells (hDPCs), outer root sheath cells (hORSCs), and on ex vivo human hair organ culture. SA significantly prolonged anagen hair growth in the in vivo mouse model. We confirmed expression of the MR in human HFs, and that SA increased the proliferation of hDPCs and hORSCs. It was found that SA enhanced hair shaft elongation in an ex vivo human hair organ culture. SA treatment of hDPCs led to increased c-myc, hepatocyte growth factor, keratinocyte growth factor and vascular endothelial growth factor levels and upregulation of p38 MAPK and cAMP response element-binding protein levels. Our results show that SA promotes hair growth and may serve as a new therapeutic agent in the treatment of alopecia.

Skin equivalent assay: An optimized method for testing for hair growth reconstitution capacity of epidermal and dermal cells.

Hair follicle reconstitution requires highly organized epithelial-mesenchymal interactions. Skin equivalents containing the epidermal and dermal cells with hair reconstitution capacity can reproduce these processes, but have not been established. This study was conducted to develop a hair follicle-producing three-dimensional (3D) skin equivalent assay using neonate mouse epidermal and dermal cells. A skin equivalent comprised of mouse dermal cells (MDCs) embedded in type I collagen and overlaid with mouse epidermal cells (MECs) was used. MDCs were mixed with type I collagen and cultured for 7 days. One day after adding MECs on top, the composites were grafted onto nude mice. MDCs cultured on a two-dimensional (2D) plate for 7 days and mixed with MECs as a negative control, and freshly isolated MDCs and MECs mixture (chamber assay) as a positive control were also grafted. Six weeks after grafting, regenerated hair follicles were analysed. Our 3D skin equivalent culture assay reproducibly regenerated hair follicles, while MDCs precultured in the 2D model with MECs did not. Compared to the chamber assay, which produced randomly oriented hair follicles, nearly all regenerated hair follicles in our assay extruded through the skin and numerous regenerated hair follicles were higher than those in the chamber assay. Several representative genes associated with hair induction showed higher expression in our assay than in the 2D model. When Wnt3a was added, the number of regenerated hairs increased. Organized hair follicle regeneration was accomplished using our assay. This approach can be applied to assess a test agent with hair growth-promoting effects.

Enhancement of Human Hair Growth Using Ecklonia cava Polyphenols.

BACKGROUND: Ecklonia cava is a brown alga that contains various compounds, including carotenoids, fucoidans, and phlorotannins. E. cava polyphenols (ECPs) are known to increase fibroblast survival. The human dermal papilla cell (hDPC) has the properties of mesenchymal-origin fibroblasts. OBJECTIVE: This study aims to investigate the effect of ECPs on human hair growth promotion in vitro and ex vivo. METHODS: MTT assays were conducted to examine the effect of ECPs on hDPC proliferation. Hair growth was measured using ex-vivo hair follicle cultures. Real-time polymerase chain reaction was performed to evaluate the mRNA expression of various growth factors in ECP-treated hDPCs. RESULTS: Treatment with 10 µg/ml purified polyphenols from E. cava (PPE) enhanced the proliferation of hDPCs 30.3% more than in the negative control (p<0.001). Furthermore, 0.1 µg/ml PPE extended the human hair shaft 30.8% longer than the negative control over 9 days (p<0.05). Insulin-like growth factor-1 (IGF-1) mRNA expression increased 3.2-fold in hDPCs following treatment with 6 µg/ml PPE (p<0.05). Vascular endothelial growth factor (VEGF) mRNA expression was also increased 2.0-fold by 3 µg/ml PPE (p<0.05). Treatment with 10 µg/ml PPE reduced oxidative stress in hDPCs (p<0.05). CONCLUSION: These results suggest that PPE could enhance human hair growth. This can be explained by hDPC proliferation coupled with increases in growth factors such as IGF-1 and VEGF. Reducing oxidative stress is also thought to help increase hDPCs. These favorable results suggest that PPE is a promising therapeutic candidate for hair loss.

Role of Arachidonic Acid in Promoting Hair Growth.

BACKGROUND: Arachidonic acid (AA) is an omega-6 polyunsaturated fatty acid present in all mammalian cell membranes, and involved in the regulation of many cellular processes, including cell survival, angiogenesis, and mitogenesis. The dermal papilla, composed of specialized fibroblasts located in the bulb of the hair follicle, contributes to the control of hair growth and the hair cycle. OBJECTIVE: This study investigated the effect of AA on hair growth by using in vivo and in vitro models. METHODS: The effect of AA on human dermal papilla cells (hDPCs) and hair shaft elongation was evaluated by MTT assay and hair follicle organ culture, respectively. The expression of various growth and survival factors in hDPCs were investigated by western blot or immunohistochemistry. The ability of AA to induce and prolong anagen phase in C57BL/6 mice was analyzed. RESULTS: AA was found to enhance the viability of hDPCs and promote the expression of several factors responsible for hair growth, including fibroblast growth factor-7 (FGF-7) and FGF-10. Western blotting identified the role of AA in the phosphorylation of various transcription factors (ERK, CREB, and AKT) and increased expression of Bcl-2 in hDPCs. In addition, AA significantly promoted hair shaft elongation, with increased proliferation of matrix keratinocytes, during ex vivo hair follicle culture. It was also found to promote hair growth by induction and prolongation of anagen phase in telogen-stage C57BL/6 mice. CONCLUSION: This study concludes that AA plays a role in promoting hair growth by increasing the expression of growth factors in hDPCs and enhancing follicle proliferation and survival.

Role of nuclear factor E2-related factor 2 (Nrf2) in epidermal differentiation.

Nuclear factor E2-related factor 2 (Nrf2) is one of the most important redox-sensitive transcription factors regulating expression of antioxidative genes and cytoprotective enzymes, which constitute the cellular response to oxidative stress and xenobiotic damage. In this study, we investigated the functional role of Nrf2 during normal epidermal keratinocyte (NHEK) differentiation. Immunohistochemical staining showed that Nrf2 is expressed from basal to granular layer of epidermis. When cultured NHEKs were treated with 1.2 mM calcium, Nrf2 expression was increased gradually in protein levels and Nrf2 translocated into the nucleus in a differentiation-dependent manner. When Nrf2 was overexpressed in NHEK by adenoviral transduction, the expression of the NHEK differentiation marker loricrin and keratin 10 was increased and overexpression of Nrf2 also increased the luciferase activity of loricrin in the absence of calcium. These results suggest that Nrf2 helps to promote the differentiation of epidermal keratinocytes.

S100A8 and S100A9 are messengers in the crosstalk between epidermis and dermis modulating a psoriatic milieu in human skin.

S100A8 and S100A9 are members of the S100A8 protein family that exist as homodimers and heterodimers in neutrophils, monocytes, and macrophages. Recent studies have shown the pivotal roles of S100A8 and S100A9 in the propagation of inflammation and keratinocyte proliferation in psoriasis. We found significant up-regulation of S100A8 and S100A9 secretion from keratinocytes in psoriatic lesions. To mimic the in vivo secretory conditions of S100A8 and S100A9 from psoriatic epidermal keratinocytes, we used the culture medium (CM) of S100A8 and S100A8/A9 adenovirus-transduced keratinocytes to investigate the functions of S100A8 and S100A9. We detected increased levels of various pro-inflammatory cytokines in the CM, including IL-8 and TNF-α, which are involved in aggravating psoriatic skin lesions, and IL-6 and members of the CXCL family of pro-angiogenic cytokines. The CM increased immune cell migration and increased angiogenesis in human umbilical vein endothelial cells. In conclusion, we found that the upregulated production of S100A8 and S100A9 by psoriatic epidermal keratinocytes activated adjacent keratinocytes to produce several cytokines. Moreover, S100A8 and S100A9 themselves function as pro-angiogenic and chemotactic factors, generating a psoriatic milieu in skin.

Silver nanoparticles modify VEGF signaling pathway and mucus hypersecretion in allergic airway inflammation.

The anti-inflammatory action of silver nanoparticles (NPs) has been reported in a murine model of asthma in a previous study. But more specific mechanisms of silver NPs in an attenuation of allergic airway inflammation have not yet been established. Vascular and mucous changes are believed to contribute largely in pathophysiology in asthma. Among various factors related to vascular changes, vascular endothelial growth factor (VEGF) plays a pivotal role in vascular changes in asthma. Mucin proteins MUC5AC and MUC5B have been implicated as markers of goblet cell metaplasia in lung pathologies. The aim of this study was to investigate the effects of silver NPs on VEGF signaling pathways and mucus hypersecretion. Ovalbumin (OVA)-inhaled female BALBc mice were used to evaluate the role of silver NPs and the related molecular mechanisms in allergic airway disease. In this study, with an OVA-induced murine model of allergic airway disease, it was found that the increased levels of hypoxia-inducible factor (HIF)-1α, VEGF, phosphatidylinositol-3 kinase (PI3K) and phosphorylated-Akt levels, and mucous glycoprotein expression (Muc5ac) in lung tissues were substantially decreased by the administration of silver NPs. In summary, silver NPs substantially suppressed mucus hypersecretion and PI3K/HIF-1α/VEGF signaling pathway in an allergic airway inflammation.

Attenuation of allergic airway inflammation and hyperresponsiveness in a murine model of asthma by silver nanoparticles.

The use of silver in the past demonstrated the certain antimicrobial activity, though this has been replaced by other treatments. However, nanotechnology has provided a way of producing pure silver nanoparticles, and it shows cytoprotective activities and possible pro-healing properties. But, the mechanism of silver nanoparticles remains unknown. This study was aimed to investigate the effects of silver nanoparticles on bronchial inflammation and hyperresponsiveness. We used ovalbumin (OVA)-inhaled female C57BL/6 mice to evaluate the roles of silver nanoparticles and the related molecular mechanisms in allergic airway disease. In this study with an OVA-induced murine model of allergic airway disease, we found that the increased inflammatory cells, airway hyperresponsiveness, increased levels of IL-4, IL-5, and IL-13, and the increased NF-κB levels in lungs after OVA inhalation were significantly reduced by the administration of silver nanoparticles. In addition, we have also found that the increased intracellular reactive oxygen species (ROS) levels in bronchoalveolar lavage fluid after OVA inhalation were decreased by the administration of silver nanoparticles. These results indicate that silver nanoparticles may attenuate antigen-induced airway inflammation and hyperresponsiveness. And antioxidant effect of silver nanoparticles could be one of the molecular bases in the murine model of asthma. These findings may provide a potential molecular mechanism of silver nanoparticles in preventing or treating asthma.

Role of plasminogen activator inhibitor-2 (PAI-2) in keratinocyte differentiation.

BACKGROUND: Plasminogen activator inhibitor-2 (PAI-2) is an enzyme inhibitor which is involved in various biological processes including cell differentiation, tissue regrowth and regeneration. Although PAI-2 has been originally isolated as an extracellular inhibitor of urokinase plasminogen activator (uPA), recent studies indicate that PAI-2 has other intracellular effects in keratinocyte, such as the component of cornified envelope. OBJECTIVE: The aim of this study is to investigate the expression and functional role of PAI-2 during the keratinocyte differentiation. METHODS: We transduced keratinocytes with adenovirus harboring the expression cassette for PAI-2, then examined the effect on keratinocytes differentiation. RESULTS: When cultured epidermal keratinocytes were treated with 1.2 mM calcium, PAI-2 expression was increased time-dependently at both mRNA and protein levels. The calcium-induced PAI-2 expression was abolished by treatment with p38 MAPK inhibitor, while overexpression of MKK6 led to the increase of PAI-2 expression. When PAI-2 was overexpressed by adenoviral transduction, the expression of keratinocyte differentiation markers such as involucrin, keratin 10 and loricrin was markedly increased. Concomitantly, overexpression of PAI-2 resulted in the retardation of cell growth, with the increase of Rb and p53. CONCLUSION: These results suggest that PAI-2 has a role for promoting the differentiation of epidermal keratinocytes.

Pitx2, a beta-catenin-regulated transcription factor, regulates the differentiation of outer root sheath cells cultured in vitro.

BACKGROUND: Beta-catenin exerts its crucial role in hair follicle development and hair growth cycle. Although the importance of Wnt/beta-catenin is well recognized, the downstream effectors of beta-catenin have not been clearly elucidated yet. OBJECTIVE: The aim of this study is to identify the beta-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells. METHODS: We transduced ORS cells with adenovirus harboring the expression cassette for constitutive active form of beta-catenin, then performed cDNA microarray. RESULTS: Overexpression of beta-catenin led to the upregulation of hair cell differentiation markers such as keratin 16 and 17. In addition, the expression of Pitx2, a bicoid-type homeodomain transcription factor, was also increased by overexpression of beta-catenin in ORS cells cultured in vitro. To investigate the potential role of Pitx2, we made the recombinant adenovirus expressing Pitx2, then transduced into the cultured ORS cells. Interestingly, Pitx2 induced the expression of keratin 16 and 17, indicating that Pitx2 activates ORS cells towards the follicular differentiation pathway preferentially. CONCLUSION: Our results implicate the potential importance of Pitx2 as a beta-catenin downstream modulator in hair growth control.

The role of nkx2.5 in keratinocyte differentiation.

BACKGROUND: Nkx2.5 is a homeodomain-containing nuclear transcription protein that has been associated with acute T-lymphoblastic leukemia. In addition, Nkx2.5 has an essential role in cardiomyogenesis. However, the expression of Nkx2.5 in the skin has not been investigated. OBJECTIVE: In an attempt to screen the differentially regulated genes involved in keratinocyte differentiation, using a cDNA microarray, we identified Nkx2.5 as one of the transcription factors controlling the expression of proteins associated with keratinocyte differentiation. METHODS: To investigate the expression of Nkx2.5 during keratinocyte differentiation, we used a calcium-induced keratinocyte differentiation model. RESULTS: RT-PCR and Western blot analysis revealed that the expression of Nkx2.5, in cultured human epidermal keratinocytes, increased with calcium treatment in a time-dependent manner. In normal skin tissue, the expression of Nkx2.5 was detected in the nuclei of the keratinocytes in all layers of the epidermis except the basal layer by immunohistochemistry. In addition, the expression of Nkx2.5 was significantly increased in psoriasis and squamous cell carcinoma, but was barely detected in atopic dermatitis and basal cell carcinoma. CONCLUSION: These results suggest that Nkx2.5 may play a role in the change from proliferation to differentiation of keratinocytes and in the pathogenesis of skin disease with aberrant keratinocyte differentiation.

Anti-apoptotic role of S100A8 in X-ray irradiated keratinocytes.

BACKGROUND: Ionizing radiation is used to treat a lot of cancers, however, it also produced unwanted side effect on normal tissues, such as radiodermatitis. We previously established an animal model for radiodermatitis, and identified many of radiation-induced genes by cDNA microarray. Of the candidates, we chose S100A8 gene for a further study. OBJECTIVE: The aim of this study is to investigate the functional role of S100A8 in X-ray irradiated keratinocytes. METHODS: RT-PCR and immunohistochemistry were performed to demonstrate the S100A8 induction by X-ray irradiation. HaCaT keratinocytes were transduced with the recombinant adenovirus expressing GFP-S100A8, and then effects on cell cycle and apoptosis were analyzed using flow cytometry and Western blot. RESULTS: X-ray irradiation markedly induced S100A8 expression in the hyperplastic epidermis of mouse. Overexpression of S100A8 by adenoviral transduction led to the enhancement of cell proliferation in the absence and/or presence of X-ray irradiation, as compared with Ad/GFP control group. Furthermore, overexpression of S100A8 significantly protected the X-ray-induced apoptosis. CONCLUSION: These results suggest that S100A8 have an anti-apoptotic role in X-ray irradiated keratinocytes.